Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Abstract

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

Figure 1.

Analysis of EST databases. (A) Relative abundance of ESTs encoding cell wall–modifying enzymes and proteins taken from ~345,000 barley ESTs. Numbers indicate the actual number of EST entries for each class of enzyme or protein. CesA indicates cellulose synthase, Gsl indicates glucan synthase–like proteins, Ara/Xyl indicates α-l-arabinofuranosidases and β-d-xylosidases, and AXAH indicates arabinoxylan arabinofuranohydrolases. (B) Relative abundance of ESTs encoding XTHs in the barley EST databases. The five most abundantly represented genes are designated HvXTH1 (equivalent to PM2; Smith et al. 1997), HvXTH2 (equivalent to PM5; Schünmann et al. 1997), HvXTH3 (equivalent to XEA; Schünmann et al. 1997), HvXTH4 (equivalent to XEB; Schünmann et al. 1997), and HvXTH5 (equivalent to EXT; Smith et al. 1997). The unnamed relatively low abundance ESTs are likely to arise from nine separate genes.

Figure 2.

Phylogenetic analysis of genes encoding family GH16 glycosyl hydrolases. The phylogenetic tree is constructed from a selection of 57 members of family GH16 glycosyl hydrolases (Courtinho and Henrissat 1999). Species names and descriptions of catalytic functions together with GenBank/EMBL, SWISS-PROT, or PDB accession numbers are given for each enzyme. The enzymes can be divided into five groups, which fall within the two subfamilies GH16a and GH16b. Subfamily GH16a includes “nonspecific” (1,3/1,3;1,4)-β-d-glucan endohydrolases (boxed in blue), (1,3)-β-galactanases (boxed in green), and (1,4)-β-galactanases/κ-carrageenases (boxed in deep blue). Subfamily GH16b includes (1,3;1,4)-β-d-glucan endohydrolases (boxed in orange) and higher plant XTHs (boxed in red). The available 3D structures are shaded in red, and the modeled 3D protein structures are colored in blue. The phylogenetic trees were generated with TreePuzzle (Schmidt et al. 2002).

Figure 3.

Multiple sequence alignments of the modeled (target) sequences and the 3D structures used as templates. The enzymes of family GH16 are divided into the two subfamilies GH16a (A) and 16b (B). The numbers above the sequences in A and B indicate amino acid residues of 3D structures of the (1,4)-β-galactanase/κ-carrageenase from P. carragenovorans (Michel et al. 2001) and of the (1,3;1,4)-β-d-glucan endohydrolase from B. macerans (Hahn et al. 1995), respectively. Multiple sequence alignments of amino acid residues in the indicated regions show the difference between subfamily GH16a enzymes, comprising the (1,4)-β-galactanases/κ-carrageenases and the “nonspecific” (1,3/1,3;1,4)-β-d-glucanase subgroups. Subfamily GH16b contains the XTHs and the (1,3;1,4)-β-d-glucanases. In subfamily GH16a four amino acid residues separate the catalytic Glu residues (colored in red), while in subfamily GH16b three amino acid residues separate the catalytic Glu residues. Conserved hydrophobic residues (yellow), residues potentially involved in catalytic amino acid function (blue), and the conserved aromatic platform amino acid residues (green) are also indicated. The positions of these amino acid residues in the 3D structures and molecular models are shown in Figures 4 ▶ and 5 ▶. Putative Ca²⁺-binding amino acid residues (aqua blue) and nonaromatic substrate binding residues (orange) are also shown. The PYX motif that is inserted in the XTHs is shaded in gray/yellow in B. The multiple sequence alignments were calculated by using ClustalW (Thompson et al. 1994) with the following sequences: P. carrageenovora (PDB entry 1DYP), Z. galactanivorans (Swis-Prot: AAF21820), S. purpuratus (Swis-Prot: Q26660), B. circulans (Swis-Prot: P23903), B. macerans (PDB entry 1BYH), B. lichiformis (PDB entry 1GBG), B. brevis (Swis-Prot: P37073), C. thermocellum (P29716), V. labrusca (NCBI: BAB78506), and V. angularis (Swis-Prot: A49539).

Figure 4.

Molecular surface representations of family GH16 and GH11 enzymes. Family GH16a members include the 3D structure of the P. carragenovora (1,4)-β-galactanase/κ-carrageenase (PDB entry 1DYP) (A), and the model of the S. purpuratus nonspecific (1,3/1,3;1,4)-β-d-glucanase (Swis-Prot: Q26660) (B). The family GH16b members include the 3D structure of the B. macerans (1,3;1,4)-β-d-glucanase (PDB entry 1BYH) (C) and the models of the V. angularis XTH (Swis-Prot: A49539) (D) and V. labrusca XTH (NCBI: BAB78506) (E). Family GH11 is represented by the A. niger (1,4)-β-d-xylan endohydrolase (PDB entry 1UKR) (F). The catalytic amino acid residues are colored in red, amino acid residues affecting the function of catalytic amino acid residues are blue, aromatic amino acid residues are in yellow, and the aromatic platform residues (Tyr, Trp, or Phe) are highlighted in green. The figure was generated by using the PyMol program (http://www.pymol.org).

Figure 5.

Comparison of the 3D dispositions of amino acid residues in the active site regions of enzymes from families GH16 and GH11. Active site amino acid residues from the 3D structure of the family GH16 (1,3;1,4)-β-d-glucanase from B. macerans (PDB entry 1BYH) (A) are compared with those from the 3D structure of family GH16 XTH of P. tremula × tremuloides XTH (PDB entry 1UN1) (B), and with those from the 3D structure of the family GH11 (1,4)-β-d-xylan endohydrolase from A. niger (PDB entry 1UKR) (C). The catalytic amino acid residues are shown in red, and the Asp residue between the two catalytic residues (Fig. 3 ▶) is shown in cyan. The distinguishing aromatic amino acid residues are colored in green, and the aromatic residues that might be involved in substrate binding are colored in yellow. In all but the two catalytic residues, the nitrogen and oxygen atoms are colored in blue and in red, respectively. Red circles show the conserved “aromatic triad.” The figure was generated by using the PyMol program (http://www.pymol.org).